Son et al. Journal of Ecology and Environment (2019) 43:3 Journal of Ecology https://doi.org/10.1186/s41610-018-0102-3 and Environment

SHORTCOMMUNICATION Open Access Pollination of Cleisostoma scolopendrifolium (Orchidaceae) by megachilid bees and determinants of fruit set in southern Hyun-Deok Son1, Hyoung-Tak Im2 and Sei-Woong Choi3*

Abstract We investigated the pollinators of Centipede’sfootorchid(Cleisostoma scolopendrifolium) at five locations in southern Korea. Only one species of megachilid bee (Megachile yasumatsui) was observed at three of the sites (, Haenam, and Wando). We assumed that the megachilid bee worked as a sole pollinator of C. scolopendrifolium based on the visiting behavior of the bees the attached pollinia. Fruits were observed at the three locations that bees visited: 74% fruit set at Mokpo, 59% at Wando, and 31% at Haenam. By contrast, at two of the locations where megachilid bees did not visit the plants ( and Jindo), there were no fruits set. The differences in fruit set rates at the three orchid populations where bees were observed appear to be related to bee abundance and flower visitation rate rather than to differences in flower abundance. The pollination interaction between C. scolopendrifolium and megachilid bees appears to involve sexual deception since only male bees were attracted to the flowers. The underlying mechanism involved in this interaction needs investigation. Keywords: Pollinator, Cleisostoma scolopendrifolium, Megachilid bee, Fruit set, Korean peninsula

Background pollinia (pollen masses) on male bees, and lack additional The pollination ecology of orchid (Orchidaceae) has floral rewards such as nectar, pollen, or pseudopollen. attracted the attention of many scientists including In non-autogamous orchids, a single season pollinator Charles Darwin. Although the majority of pollination limitation to fruit setting is strongly indicated by a low network between animals and plants involves food re- natural fruit set rate reflected by an extraordinarily high wards such as nectar and pollen to the pollinators, or- proportion of flowering individuals that do not set fruit, chids are exceptional in that as many as one third of presumably because of a lack of pollinators (Darwin the 30,000 or so species achieve pollination by decep- 1877). For example, Tolumnia variegate, a small twig tion; for example, they may lure pollinators to the epiphyte in the West Indies has an extremely low nat- flower by using a food deceptive, advertising the pres- ural fruit set rate, often below 5% (Ackerman and Oliver ence of food by displaying bright colors and sweet scents 1985). However, hand pollination results in a marked in- (Jersakova et al. 2006;Ledford2007). In addition, they can crease in fruit set, to over 70% (Calvo 1993). Similarly, use sexual deception, as more than 600 species of orchids Disa tenuifolia in South Africa depends on the exploit- have evolved adaptations for pollination by male euglos- ation of instinctive foraging and territorial behavior of sines (Dodson et al. 1969). Euglossine-pollinated orchids male megachilid bees and its fruiting success is pollin- produce attractive volatile compounds (terpenes and aro- ator dependent (Johnson and Steiner 1994). matics), exhibit intricate mechanisms for the attachment of Over 100 species of Cleisostoma have been reported in tropical and subtropical Asia and Australia (Wood * Correspondence: [email protected] 2014). Cleisostoma scolopendrifolium, an epiphyte or- 3Department of Environmental Education, Mokpo National University, 1666 chid that grows on rocks, has been recorded on sub- Youngsan-ro, Chungkye-myon, Muan, Jeonnam 58554, South Korea tropical and temperate trees of Northeast Asia, Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Son et al. Journal of Ecology and Environment (2019) 43:3 Page 2 of 5

including Korea, , and . The extent of occur- pre-study observations of the sites (Son et al. 2017), we rence of this species in Korea has been estimated to be selected the peak time of insect visits (between 9:00 and 2000 km2 (National Institute of Biological Resources 11:00 a.m.) as the time to record insect visits. The visit- 2014). Each year, C. scolopendrifolium plants produce a ing behaviors of the bee were classified into two types: new rhizome that ends in a vegetative shoot composed of “searching” and “staying.” If a bee approached to a 6–10-mm-long leaves. The inflorescence is a 1- or 2-flow- flower but only moved around and did not land, the be- ered raceme, located laterally, and often shorter than havior was recorded as “searching.” If a bee landed on a leaves. In Korea, most C. scolopendrifolium flower late in flower and stayed on that flower for more 10 s, the be- July. This orchid species is self-incompatible and insect havior was recorded as “staying” (Table 2). pollinated. The pollinator species of C. scolopendrifolium in Mokpo, South Korea, is assumed to be male megachilid Determination of fruit set bees, based on the observation that only male megachilid To estimate fruit sets, we monitored 1.0 m2 quadrats at bees have visited the orchid and the occurrence of pollinia each C. scolopendrifolium sampling site during the attached to the head of flower-visiting megachilid bees period in which the plant flowered and set fruit. We (Son et al. 2017). counted flowers of orchid populations between 19 July There is a large body of literature describing the pol- 2018 and 26 July 2018 and counted fruits between 11 lination biology of orchids worldwide. However, there August 2018 and 15 August 2018. The fruit set rate was is little information on the pollination ecology of or- measured by calculating the fruit to flower ratio in each chid species in Korea. This study aimed to confirm quadrat. that male megachilid bees are the unique pollinators of C. scolopendrifolium in southern Korea and to der- Results termine whether pollinator visitation affects the fruit- Is the male megachilid bee the special pollinator of C. ing of the orchid. scolopendrifolium? Only male Megachile yasumatsui were observed visiting Materials and methods C. scolopendrifolium flowers at the MP, HN, and WD Study sites sites. Megachilid bees searched 68 flowers and stayed on In this study, we included five separate populations of 19 flowers at MP, searched 28 flowers and stayed on 6 Cleisostoma scolopendrifolium, all located in the south- flowers at WD and searched 11 flowers and stayed on 3 ern part of the Korean peninsula: Mt. Yudalsan in the flowers at HN. Orchid pollinia were observed to be at- city of Mokpo (MP), Mt. Galdusan of tached to the heads of megachilid bees that searched (HN), Gunoe-myeon of (WD), near Dado and stayed on the orchid flowers at MP, HN, and WD reservoir close to Naju city (NJ) and Mt. Geumgolsan of (Fig. 1). No bee searched or stayed on the orchid flowers (JD) and all in the province of Jeollanamdo at JD. On one occasion, a honey bee was observed at NJ. (Table 1). These five sites were geographically separated. No orchid pollinia were attached to the body of the We selected sites that had orchid plants present over an honey bee at NJ. area of at least 1.0 m2 because small areas with sparsely occurring flowers may affect pollinator visit frequency Do visit frequencies of pollinator enhance fruit set? (Dauber et al. 2010). Fruit setting occurred at each of the three sites where male megachilid bees visited C. scolopendrifolium plants. Pollinators Fruit set rates were 74% (111 fruits among 151 flowers) We observed the insect visitors to each of the five separ- at MP, 59% (366 fruits among 620 flowers) at WD, and ate C. scolopendrifolium sites. To estimate flower-visiting 31% (171 fruits among 547 flowers) at HN. The C. frequencies, the numbers of insects that visited flowers in each quadrat at each site were counted. Based on Table 2 Fruit set rates and bee visit behavior at five Cleisostoma Table 1 Observation sites of Cleisostoma scolopendrifolium orchid scolopendrifolium sites. For site information, see Table 1 in southern South Korea Site No. of No. of Fruit set Visitor No. of flower visited flowers fruits (%) Site Location Patch size species Searching Staying 2 MP Mt. Yudalsan, Mokpo City, Jeollanamdo 2 m MP 151 111 74 Megachile yasumatsui 67 16 WD Sinhak-ri, Wando County, Jeollanamdo 2 m2 WD 620 366 59 Megachile yasumatsui 28 6 NJ Near Dado Reservoir, Naju City, Jeollanamdo 1600 m2 HN 547 171 31 Megachile yasumatsui 11 3 HN Mt. Galdusan, Haenam County, Jeollanamdo 1.5 m2 NJ 488 0 0 Apis mellifera 11 JD Mt. Geumgolsan, Jindo County, Jeollanamdo Distributed by patch JD 166 0 0 ––– Son et al. Journal of Ecology and Environment (2019) 43:3 Page 3 of 5

Fig. 1 Life history of Cleisostoma scolopendrifolium and male megachilid bee pollinator in southern South Korea. a.Orchidpopulationat Wando. b. Pollinia matured. c. After pollination. d.Fruit.e. Pollinia attached to the bee head. f. Megachilid bee pollinator visitation scolopendrifolium plants observed at NJ and JD, where appendiculata and Cymbidium goeringii showed low no megachilid bees were observed, failed to produce fruit set rates in natural condition (1.3–2.0% and 0.4– fruit (Table 2). 0.6%, respectively), but artificial self-pollination and geitonogamous pollination produced fruit set rates of 95.7% and 96.6%, respectively, for C. appendiculata, Discussion and 95.5% and 95.6%, respectively, for C. goeringii. Based on our observation, the specific pollinators of The extremely low fruit set rates observed in these two or- Korean C. scolopendrifolium orchid are male megachi- chid species under natural conditions has been explained lid bees (Megachile yasumatsui), and the fruit produc- as the result of pollinator limitation (Chung 2004). tion process between this bee and C. scolopendrifolium Fruit sets were present at three of the orchid observa- is assumed to be a specialized one. Mendel (1995)sug- tion sites (MP, WD, and HN), but there was a wide range gested that fruit set in outcrossing orchids in their natural in fruit set rates. The patch sizes of the three observed or- habitat is generally limited by pollinator availability. Pol- chid sites were similar (1.5–2m2), but the numbers of linator visits to flowers enhance subsequent fruiting and flowers at WD (620) and HN (547) were notably higher enable outcrossing orchid populations to live longer. We than that at MP (151). Flower searching behavior of mega- observed a wide range of fruit set rates (31% to 74%) in chilid bees was most commonly observed at MP (67 three locations (MP, WD, and HN) and no fruit sets at searches) followed by that at WD (28 searches) and HN two orchid locations (NJ and JD) where no megachilid (11 searches). However, the proportion of bees showing bees were observed. The frequency of visits by poten- flower staying after searching at these three locations were tial pollinators is low in many orchids (Darwin 1877; not notably different: 23.9% (MP), 27.3% (HN), and 21.4% Ackerman 1981) and is the main cause of low fruit set (WD), respectively. The differences in fruit set rates at the rates. An experiment using hand pollination of orchids three orchid locations where bees were observed appear showed higher fruit set ratesthaninopenpollination to be related to bee abundance and flower visitation rate (Calvo 1993). For example, the orchids Cremastra rather than to differences in flower abundance. Son et al. Journal of Ecology and Environment (2019) 43:3 Page 4 of 5

The orchid family is exceptional for its unusually Acknowledgements high frequency of non-rewarding species compared to We thank the Cultural Heritage Administration and ministry of Environment, Korea for allowing to access and examine the endangered orchid plant. that in other plant families (Jersáková et al. 2006). Gen- erally, food deception is the most common pollination- Funding related mechanism in non-rewarding orchids (reported This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of in 38 genera) followed by sexual deception (18 genera) Science and ICT (NRF – 2017R1D1A2B03028800). (Jersáková et al. 2006). Nearly all species of the Medi- terranean orchid genus Ophrys are pollinated by means Availability of data and materials of sexual deception of the pollinator (Pouyanne 1917; The datasets used and/or analyzed during the current study are available from the corresponding author (Dr. Sei-Woong Choi, [email protected]) Kullenberg 1961; Paulus and Gack 1990). The flowers on reasonable request. of such orchids mimic the female members of the pol- linator species, usually solitary bees and wasps, and Authors’ contributions HDS and HTI observed and analyzed the pollinator’s behavior and orchid only male members are attracted to and try to copulate biology data regarding the flowering and fruiting. SWC interpreted the data with the flower’s labellum. During such “pseudocopula- set and was a major contributor in writing the manuscript. All authors read tions,” pollinia become attached to the bees and are and approved the final manuscript. transferred during further visits of the males to other Ethics approval and consent to participate flowers (Schiestl 2005). Schiestl and Cozzolino (2008) Not applicable. found that Ophrys attracts male bees by mimicking fe- male mating signals through a combination of Consent for publication Not applicable. species-specific imitation female sex pheromones and unsaturated hydrocarbons (alkenes). The flowers of C. Competing interests scolopendrifolium have very attractive bright colors, The authors declare that they have no competing interests. such as yellow, red, and white, but there is no food re- ward for the visiting pollinators due to the absence of Publisher’sNote nectar. Based on the absence of food deception, and Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. because only male bees were attracted to the flowers, the specific pollination interaction between C. scolo- Author details 1 2 pendrifolium and megachilid bees involves sexual de- Mokpo Natural History Museum, Mokpo 58699, South Korea. Department of Biological Sciences, Chonnam National University, Gwangju 61186, South ception. The underlying mechanism of this interaction Korea. 3Department of Environmental Education, Mokpo National University, needs further experiments to elucidate the chemical 1666 Youngsan-ro, Chungkye-myon, Muan, Jeonnam 58554, South Korea. ecology of the bee-flower relationship. Received: 23 November 2018 Accepted: 25 December 2018

Conclusion References In this study, we observed that pollinator visitation af- Ackerman JD. Pollination biology of Calypso bulbosa var. occidentalis (Orchidaceae): a food-deception system. Madrono. 1981;28:101–10. fected the fruit production of C. scolopendrifolium,anout- Ackerman JD, Oliver JCM. Reproductive biology of Oncidium variegatum: moon crossing orchid. Pollinator absence can result in fruit phases, pollination, and fruit set. Amer Orchid Soc Bull. 1985;4:326–9. failure while a high number of pollinator visits can result Calvo RN. Evolutionary demography of orchids: intensity and frequency of pollination and the cost of fruiting. Ecology. 1993;74:1033–42. in a high production of fruit. Based on the zero fruiting Chung MY. Population genetics of several orchid and tree species in Korea. rate at the NJ and JD sites, where the orchid population Implications for conservation and ecology. Jinju: Gyeongsang National sizes were large, it is expected that the genetic diversity of University; 2004. Darwin C. The various contrivances by which orchids are fertilised by insects those orchid populations will decline rapidly unless there 1984 edition. Chicago: University of Chicago Press; 1877. is an increase in visits by legitimate pollinator. In a similar Dauber J, Biesmeijer JC, Gabriel WE, Kunin E, Lamborn B, et al. Effect of patch size situation, the Saitama Primula is in danger of species ex- and density on flower visitation and seed set of wild plants: a pan-European approach. J Ecol. 2010;98:188–196. tinction because the specific pollinator is reported to be Dodson CH, Dressler RL, Hills HG, Adams RM, Williams NH. Biologically active absent (Yoshida et al. 2009). Therefore, understanding the compounds in orchid fragrance. Science. 1969;164:1243–9. pollinator’s population size and pollination behaviors are Jersáková JS, Johnson D, Kindlmann P. Mechanisms and evolution of deceptive pollination in orchids. Biol Rev. 2006;81:219–35. of fundamental importance for the conservation of C. sco- Johnson SD, Steiner KE. Pollination by megachilid bees and determinants of fruit- lopendrifolium in Korea. set in the Cape orchid Disa tenuifolia. Nord J Bot. 1994;14:481–6. Kullenberg B. Studies in Ophrys pollination. Zool Bidrag Uppsala. 1961;34:1–340. Ledford H. The flower of seduction. Nature. 2007;445:816–7. Abbreviations Mendel G. The genetics of orchid pollination. In: Cingel NAVD, editor. An atlas of HN: Haenam County, Jeollanamdo, South Korea; JD: Jindo County, Jeollanamdo, orchid pollination. European orchid: Brookfield: A.A. Balkema publishers; 1995. South Korea; MP: City of Mokpo, Jeollanamdo, South Korea; NJ: City of p. 47–52. Naju, Jeollanamdo, South Korea; WD: Wando County, Jeollanamdo, South National Institute of Biological Resources. Korean red list of threatened species. Korea 2nd ed. Incheon: National Institute of Biological Resources; 2014. Son et al. Journal of Ecology and Environment (2019) 43:3 Page 5 of 5

Paulus HF, Gack C. Pollinators as prepollinating isolation factors: evolution and speciation in Ophrys (Orchidaceae). Isr J Bot. 1990;39:43–79. Pouyanne A. La fecundation des Ophrys par les insectes. Hist Nat Afr Nord. 1917; 8:6–7. Schiestl FP. On the success of swindle: pollination by deception in orchids. Naturwissenschaften. 2005;92:255–64. Schiestl FP, Cozzolino S. Evolution of sexual mimicry in the orchid subtribe orchidinae: the role of preadaptation in the attraction of male bees as pollinators. BMC Evol Biol. 2008;8:27. https://doi.org/10.1186/1471-2148-8-27. Son HD, Im HT, Choi SW. Study on the pollinator and pollination mechanism of an endangered orchid species, Sarcanthus scolopendrifolius Makino (Orchidaceae) in Korea. J Apic. 2017;32:199–203. Wood J. Cleisostoma: Distribution. In: Pridgeon AM, Cribb PJ, Chase MW, Rasmussen F, editors. Genera Orchidacearum: volume 6: Epidendroideae (part three). Oxford: Oxford University Press; 2014. Yoshida Y, Honjo M, Kitamoto N, Ohsawa R. Reconsideration for conservation units of wild Primula sieboldii in Japan based on adaptive diversity and molecular genetic diversity. Genet Res. 2009;91:225–35.